La peau est un organe sensoriel qui permet notamment à l’organisme de s’adapter à son environnement. Ainsi, de nombreuses fibres nerveuses sont présentes au sein de cette peau permettant de détecter différents stimuli tels que la pression, la douleur ou encore une variation de température. A la suite d’une brûlure profonde, ces terminaisons nerveuses sont détruites et la repousse de ces fibres lors du processus de cicatrisation est inadéquate conduisant à des handicaps souvent sérieux chez les patients. Dans un premier travail, la réalisation d’une brûlure chez des animaux présentant ou non une neuropathie périphérique induite par la résinifératoxine a permis d’étudier le rôle de l’innervation lors de la cicatrisation. Chez les animaux traités, une cicatrisation plus lente et un défaut de réinnervation par rapport aux animaux contrôles étaient observés. Pour approfondir le rôle de l’innervation dans la cicatrisation, des études in vitro ont ensuite été réalisées afin d’évaluer les interactions possibles entre les cellules neuronales et les fibroblastes dermiques. Même en l’absence de contacts directs, il a été montré que les cellules neuronales sont capables d’induire la différenciation des fibroblastes en myofibroblastes. Au vue de l’implication de l’innervation sur la différenciation des fibroblastes et sur la cicatrisation, il semble important de pouvoir proposer aux patients brûlés, pour leur traitement, un nouveau concept de substitut cutané favorisant une repousse axonale fonctionnelle au sein du tissu cicatriciel. / The skin is a sensitive organ which notably allows the body to adapt to its environment. Many nerve fibers are present in the skin and are implicated in the detection of various stimuli such as pressure, pain or temperature variation. After a deep burn injury, these nerve fibers are destroyed and their regrowth during the wound healing process is imperfect which induces serious disabilities for patients. In a first study, a burn model was developed on animals presenting or not a peripheral neuropathy induced with resiniferatoxin and had allowed to study the role of sensory innervation on wound healing. On animals treated with resiniferatoxin, wound healing was delayed and nerve regeneration was imperfect compared to control animals. To further investigate the role of innervation in wound healing, in vitro studies were then performed to evaluate possible interactions between neuronal cells and dermal fibroblasts. Even in the absence of direct contacts, it has been shown that neuronal cells are able to induce the differentiation of fibroblasts into myofibroblasts. Considering the involvement of innervation on the differentiation of fibroblasts and on wound healing, it seems important to provide burned patients with a new concept of skin substitute promoting functional axonal regrowth in the scar tissue.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Sugar is one of the most widely used products in the treatment of wounds in veterinary medicine. Its main advantage is the hygroscopic effect on tissues and the bacterial death by plasmolysis, making it a bactericidal agent due to the physical effect observed, without leading to bacterial resistance as it might occur with antibiotic therapy. The objective of this experiment was to compare cicatricial evolution in cutaneous wounds with the topical use of sugar both granulated and in the form of a gel. For such, 16 canine wounds were treated, where eight received treatment with granulated sugar (group A) and eight with sugar gel (group G). Evaluations such as mensurations of the wounded areas, bacteriological cultures and observation of the aspect of the lesions were done weekly from the moment of the first examination until the formation of enough granulation tissue to suspend the use of the products. When comparing the decline in the area between groups no statistical difference was observed at any time. However, when the areas were analyzed in each group's interval, it was possible to notice a statistically significant decline between days 1-14 and 7-14 in the group treated with sugar. In the group treated with the gel, beside those intervals, a significant decline was also noticed between days 1-7. In terms of the frequency of negative bacteriological culture, there was no statistical difference between groups, not even in the different moments of each group. The time taken for the granulation tissue to be fully formed varied from 7 to 14 days in both groups. As to applicability, the gel showed better adhesion to the wounds and filling of the product subcutaneously more effectively than the granulated sugar. The conclusion reached is that both the sugar and the gel were effective in healing the animals' wounds in this experiment, the gel having demonstrated precocity in cicatricial retraction in the first seven days. / O açúcar é um dos produtos mais utilizados para tratamento de feridas em medicina veterinária. Sua principal vantagem é o efeito higroscópico nos tecidos e morte das bactérias por plasmólise, tornando-o um bactericida pelo efeito físico realizado, sem levar à resistência bacteriana, como poderia ocorrer na terapia antibiótica. O objetivo desse experimento foi comparar a evolução cicatricial de feridas cutâneas com o uso tópico de açúcar na forma granulada e na forma de gel. Para tal, foram tratadas 16 feridas de cães, onde oito receberam tratamento com açúcar granulado (grupo A) e oito com gel de açúcar (grupo G). Avaliações como mensurações das áreas das feridas, culturas bacteriológicas e observação quanto ao aspecto das lesões foram realizadas semanalmente desde o momento do atendimento até a formação de tecido de granulação suficiente para suspender o uso dos produtos. Na comparação da diminuição da área entre os grupos não se observou diferença estatística em nenhum momento. Entretanto, quando as áreas foram analisadas nos intervalos de cada grupo, pôde-se perceber diminuição estatisticamente significativa entre os dias 1 e 14 e 7 e 14 no grupo tratado com açúcar. No grupo tratado com gel, além desses intervalos, também se percebeu diminuição significativa entre os dias 1 e 7. Com relação a frequência de cultura bacteriológica negativa, não houve diferença estatística entre os grupos, nem nos diferentes momentos de cada grupo. O tempo para completa formação de tecido de granulação variou entre sete e 14 dias em ambos os grupos. Quanto à aplicabilidade, o gel demonstrou melhor adesão nas feridas e preenchimento do produto no subcutâneo de forma mais efetiva que o açúcar granulado. Conclui-se que tanto o gel quanto o açúcar foram efetivos na cicatrização das feridas dos animais desse experimento, sendo que o gel demonstrou precocidade na retração cicatricial nos primeiros sete dias.

Elastin-like peptides (ELPs) are stimulus-responsive protein-based polymers which are attractive material for biomedical research due to their biocompatibility and unique properties. The physical properties of ELPs are dependent on the chain length and the chosen amino acid at the guest residue position. This imparts unlimited flexibility in designing ELP based biomaterials with the desired physical properties. We have shown that in addition to their physical properties, ELPs have biological activities that are conducive to tissue regeneration. Specifically, we found that ELPs induce fibroblast proliferation via cell surface heparan sulfate proteoglycans (HSPG). Furthermore, our data suggests that ELP based materials with differential proliferative potential can be designed by controlling the interaction of ELPs with HSPGs by incorporating either hydrophobic or positively charged residues within the ELP sequence. Fibroblast proliferation is important for granulation tissue formation which is important in chronic wounds as well as in healing of other tissues. The customizable biological activity of ELPs coupled with their unique physical properties will enable us to design novel, sustainable and cost effective therapies for different tissue regeneration applications. ELPs can be genetically fused to biologically active peptides or proteins. These fusions can be expressed and readily purified since they maintain the phase transitioning property of the fused ELP domain. Moreover, depending on the ELP sequence chosen the chimeric fusion sequences can self-assemble into unique structures such as nanoparticles. These structures can then be applied to the injury site where they not only provide unique topographical cues or structural support but also act as delivery vehicles for the fused bioactive protein. We developed a multifunctional nanoparticle that is comprised of PMP-D2-ELP fusion protein and different functional peptide ELP fusion proteins to preserve the bioactivity of the functional group with the existence of elastase. These heterogeneous particles will be beneficial for the delivery of combination therapies to solve multiple problems that often existed in chronic wound healing or other tissue regeneration process. In summary, this study adds to our understanding of the biological activity of ELP and the interaction mechanism that allow the regulation of cellular behavior. Furthermore this work also investigated the potential therapeutic application of ELP as a delivery platform for chronic wound healing.

Abstract Collagen XVIII is a basement membrane proteoglycan, which has three variant N-termini. These variants are coded by two promoters; promoter 1 directs the synthesis of a short variant and promoter 2 directs the synthesis of two longer variants, of which the middle variant is generated from the longest by splicing. The longest variant contains a cysteine-rich domain in its N-terminus, which shows homology to the frizzled receptors of the Wnt molecules and can inhibit Wnt/beta-catenin signalling in vitro. The C-terminal domain of collagen XVIII, endostatin, is an inhibitor of tumor growth and angiogenesis. Lack of collagen XVIII accelerates cutanous wound healing and wound angiogenesis. Overexpression of endostatin leads to delayed wound healing and the presence of morphologically abnormal wound capillaries. Moreover, endostatin overexpression leads to delayed formation of the wound epidermal basement membrane and impaired maturation of hemidesmosomes. Endostatin treatment decreases osteoblast proliferation in vitro. Moreover, osteoblast proliferation and mineralization of the matrix by osteoblasts are inhibited when cells are treated with endostatin together with VEGF. In vivo, lack of collagen XVIII leads to delayed formation of secondary ossification centers in mouse femurs, whereas overexpression of endostatin leads to a slower growth of bone length. However, both of these changes are transient and mild, suggesting that collagen XVIII/endostatin is not essential for skeletal development. The growth of hair follicles is delayed in the mice overexpressing endostatin. This delay in growth is preceded by an impaired hair follicle associated angiogenesis. Lack of collagen XVIII causes an accelerated onset of the first hair cycle. A similar change can be seen in mice lacking the long variants of collagen XVIII. Lack of the short variant causes mild acceleration in the catagen of the first cycle, and anagen is also significantly accelerated in these mice. The long variants were located in the bulge region, which contains the hair follicle stem cells, and in the basement membrane surrounding the dermal papilla. As it is known that several Wnt-inhibitors are upregulated in the bulge, our results suggest that the longest variant of collagen XVIII may have a role as a regulator of Wnt-signalling in hair follicles.

Abstract Neurofibromatosis type 1 (NF1) is a dominantly inherited neurocutaneous disorder caused by mutations in the NF1 gene. Common clinical manifestations associated with NF1 are neurofibromas, café-au-lait macules (CALM), axillary freckling and Lisch nodules of the iris. Other important manifestations are vasculopathy, a variety of osseous lesions, including short stature, scoliosis and pseudoarthrosis, optic gliomas and an increased risk for certain malignancies. The best characterized function of the NF1 gene is to act as a downregulator of Ras proto-oncogene signalling by accelerating the switch of active Ras-GTP into inactive Ras-GDP. The NF1 gene is considered a tumor suppressor since some malignancies may display a loss of heterozygosity or homozygotic inactivation of the gene. The present study investigated the behaviour and function of the NF1 gene during keratinocyte differentiation, wound healing and psoriasis using human epidermis and epidermal keratinocytes as a model. The NF1 protein was shown to associate with the intermediate filament network during keratinocyte differentiation both in vitro and in vivo, and it is thus suggested to play a role in the cytoskeletal re-organization or in the formation of cell adhesions. NF1 gene expression was also studied in psoriasis, in which keratinocytes are hyperproliferative and cell differentiation is altered. NF1 gene expression was downregulated in psoriatic keratinocytes both in vivo and in vitro, suggesting that the NF1 gene might have role in downregulating keratinocyte proliferation. During epidermal wound healing, NF1 gene expression was increased. However, the process of wound healing showed no apparent differences between NF1 patients and controls. Furthermore, an increased number of cells immunoreactive for active Ras-MAPK was demonstrated in vascular tissues of NF1 patients, but not in epidermal keratinocytes or dermal fibroblasts. The finding suggests that the NF1 protein functions as a Ras-GAP in some, but not all tissues.

Abstract Sepsis is a major challenge for healing responses maintaining homeostasis. Coagulation and inflammation are activated at the whole-body level, even in undamaged tissues. Despite constantly growing knowledge and advances in care, high mortality in severe sepsis remains. It was hypothesised that tissue regeneration processes may also be altered in severe sepsis. The study population consisted of 44 patients with severe sepsis and 15 healthy controls. Serum samples were obtained during ten days of severe sepsis and twice again, three months and six months later. Experimental suction blisters were performed twice during severe sepsis and at 3 and 6 months. Serum samples were obtained and suction blisters were induced once in controls. Biochemical analyses were performed to assess the level of procollagen I and III aminoterminal propeptides (PINP, PIIINP), reflecting the synthesis of corresponding collagens; in serum and suction blister fluid. In addition collagen I degradationproduct in serum was measured. Physiological measurements of transepidermal water loss and blood flow were done in order to evaluate the re-epithelisation rate and blood flow in an experimental wound. Levels of matrix metalloproteinases (MMPs) 2, 8 and 9 were measured from serum and suction blister fluid. Decrease in water evaporation from an experimental blister wound was slower in sepsis than in controls. On the fourth day the sepsis patients had higher blood flow in the blister wound than the controls (both in the healing wound and in the newly induced wound). The procollagen III aminoterminal propeptide (PIIINP) levels were increased in serum in severe sepsis, whereas procollagen I aminoterminal propeptide (PINP) levels were not, making up a pronounced PIIINP/PINP ratio. PIIINP and PINP levels were associated with disease severity and outcome. In addition, collagen I degradation measured with ICTP assay was increased in severe sepsis and PINP/ICTP ratio was lower. Furthermore, the overall protein concentration and PINP and PIIINP levels were low in suction blister fluid, which implies that the balance of the extracellular matrix consistence is disturbed in uninjured skin in severe sepsis. Then again in survivors the levels of PINP and PIIINP were up-regulated at three months but returned to normal by six months. MMP-9 levels in serum and skin blister fluid were lower in severe sepsis than in controls during the ten days studied. The MMP-2 levels were found to be increased both in serum and in skin blister fluid in severe sepsis in comparison to the controls and MMP-2 was associated with disease severity and outcome. MMP-8 was increased in serum and in skin blister fluid. In conclusion, the balance of collagen turnover is altered in severe sepsis in serum and skin and epidermal re-epithelisation is delayed. The levels of MMP-2 and MMP-8 are increased whereas levels of MMP-9 are depressed.

Le retard de cicatrisation (RETCIC) est un problème clinique majeur, touchant les gens âgés, les diabétiques, drépanocytaires (DR), ou traités par les glucocorticoides (GC). Nous avons abordé les mécanismes des ulcères DR dans un modèle transgénique ayant un gène beta globine humain muté (SAD). Les souris SAD âgées ont un RETCIC corrélé au degré d'anémie et d'hemolyse, comme dans la DR humaine ; le RETCIC est lié à l'altération de l'angiogenèse cutanée et un faible recrutement de cellules endothéliales progénitrices (EPC) depuis la moelle. La sécrétion de CXCL12 dans la plaie par les kératinocytes et les cellules inflammatoires est diminuée. Fait notable, le traitement local de la plaie par des EPC ou du CXCL12 recombinant restaurent l'angiogenèse et une cicatrisation des souris SAD âgées. Nous avons ensuite abordé le rôle du récepteur minéralocorticoide dans la cicatrisation cutanée, et proposé ses antagonistes (MRA) pour améliorer le RETCIC en pathologie (traitement local GC, diabète murin); j'ai aussi participé à la démonstration de l'effet bénéfique des MRA sur l'atrophie épidermique GC-induite chez l'homme. A l'aide d'explants de peau murine et humaine, de plaies cutanées crées sur le dos de souris, et par l'analyse de la cicatrisation de biopsies chez le volontaire sain après traitement GC (étude post-hoc de l'essai SPIREPI), nous montrons que les MRA améliorent le RETCIC pathologique, et n'affectent pas la peau saine normale. Nous avons montré que l'effet bénéfique des MRA topiques implique une augmentation de la prolifération kératinocytaire et le canal sodium épithelial. Ces résultats montrent un rôle inédit et important du MR cutané dans la cicatrisation. / Impaired wound healing is a major unsolved clinical problem in aging, diabetes, sickle cell disease (SCD), glucocorticoid (GC) therapy or Cushing syndrome.First, we investigated the mechanisms underlying SCD ulcers, using SAD transgenic mouse model with mutated human beta globin. Old SAD mice displayed delayed healing correlated with the severity of anemia and hemolysis, as in human SCD, related to impaired cutaneous angiogenesis and poor endothelial progenitor cell (EPC) mobilization from the bone marrow. CXCL12 secretion by keratinocytes and inflammatory cells in SAD wounds was low. Noticeably, local wound therapy with EPCs or with recombinant CXCL12 restored wound angiogenesis and healing in old SAD mice.Second, we questioned the role of mineralocorticoid receptor (MR) in wound healing and proposed that MR blockade could improve reepithelialization in pathological situations (GC-treated skin, diabetic mouse models); I also participated to the demonstration that MR blockade limits GC-induced epidermal atrophy in human skin. We used skin explants from mouse and humans, full thickness skin wounds on the back of mice, and analyzed wound closure after skin biopsy in healthy volunteers pre-treated with GC, as a post-hoc study of the SPIREPI clinical trial. We found that MR blockers can rescue the pathological delayed wound healing while they did not modify wound closure in normal condition. We show that the improvement of GC-induced wound healing delay by MR blockade involves restoration of keratinocyte proliferation and down-regulation of the epithelial sodium channel. These results suggest an important and entirely novel role for MR in to improve delayed wound closure.

Le diabète non insulino-dépendant de type 2 demeure un problème majeur de santé publique. Cette pathologie associée au vieillissement, à la sédentarisation et à l'obésité induit un taux élevé de complications. Parmi celles-ci, le défaut de cicatrisation et plus particulièrement le syndrome du " pied diabétique " est une des causes majeures de morbidité et de mortalité chez les patients diabétiques. La compréhension de la dérégulation des mécanismes cellulaires et moléculaires du processus cicatriciel chez le diabétique représente un enjeu essentiel pour le développement de nouvelles stratégies thérapeutiques. C'est dans ce contexte que ce travail de thèse a été réalisé. La cicatrisation des plaies est un processus physiopathologique complexe, multifactoriel et dynamique visant à rétablir l'intégrité et la fonctionnalité des tissus lésés. Cette réponse biologique nécessite une orchestration et une communication précise entre les cellules inflammatoires infiltrées et les différentes populations cellulaires des tissus. De manière conventionnelle, on distingue 4 principales phases qui peuvent se chevaucher : une première étape d'hémostase généralement couplée à une phase inflammatoire précoce et arrêtée par une phase de résolution de l'inflammation. Une période de prolifération/ régénération permet la génération du nouveau tissu, et enfin, la phase de remodelage tissulaire permet au tissu de retrouver des caractéristiques d'élasticité et de souplesse proche du tissu original. Les données de la littérature suggèrent que le retard de cicatrisation des personnes diabétiques résulterait de la chronicité de la phase inflammatoire. Notre hypothèse de travail a été de démontrer que cette dérégulation était la conséquence d'un défaut majeur de la phase de résolution de l'inflammation. Notre objectif final a été d'agir sur cette phase pour accélérer le processus cicatriciel. La première étape de ce travail de thèse a consisté à mettre en place un modèle de lésions cutanées par excision chez des souris diabétiques de type 2 (en régime hyper lipidique ou déficientes pour le récepteur de la leptine, db/db). Ce modèle expérimental, combiné à la mise au point d'un dispositif médical innovant nous a permis (i) de recueillir stérilement les cellules et l'exsudat produit au niveau de la lésion cutanée, (ii) d'évaluer les caractéristiques phénotypiques et fonctionnelles des cellules de l'exsudat et (iii) d'évaluer le taux des médiateurs pro- et anti-inflammatoires au cours du temps. L'ensemble de ce travail a pu être réalisé en préservant l'environnement du tissu cicatriciel. Cette première étape a débouché sur le dépôt de 2 brevets en décembre 2011 (FR 2984719 - FR 2984722) suivi de leur extension Européenne l'année suivante. Cette approche expérimentale nous a permis de démontrer que la dérégulation du processus cicatriciel de souris diabétiques de type II était due, à une forte infiltration au niveau de la lésion de polynucléaires neutrophiles, de macrophages inflammatoires M1 et à un défaut d'afflux de macrophages anti-inflammatoires M2. Tout cela était associé à un défaut d'apoptose des cellules inflammatoires et de leur efférocytose. Ces dérégulations signent un défaut de résolution de l'inflammation que nous avons pu valider par la mise en évidence au niveau de l'exsudat inflammatoire et du tissu cicatriciel d'un excès de leucotriène B4 (LTB4) (métabolite de l'acide arachidonique pro-inflammatoire provenant de la voie de la 5-lipoxygénase) au dépend de la lipoxine A4 (LXA4) (métabolite de l'acide arachidonique anti-inflammatoire produit par les voies des 5-LOX et 12/15-LOX). Ces résultats acquis, nous nous sommes attachés à cibler, par une approche pharmacologique originale, la phase de résolution de l'inflammation pour favoriser la fermeture des plaies des souris diabétiques. / Non-insulin dependent type 2 diabetes is a major public health problem due to its prevalence and the high rate of its degenerative complications. Among these problems, healing disorders and more particularly "diabetic foot" ulcers are one of the leading cause of morbidity and mortality of diabetic patients. The understanding of the deregulation of cellular and molecular mechanisms of the wound healing process in diabetic patients is a key issue for the development of new therapeutic strategies. This work was carried out in this context. Wound healing is a physiological multifactorial and dynamic process aiming to the restoration of the integrity and functionality of injured tissue. This complex biological response requires an orchestration and a precise communication between immuno-inflammatory cells and resident cells in the wound tissue. This process can be divided into four distinct but overlapping phases: an inflammatory phase involving the initial stage of hemostasis arrested by a resolution phase of inflammation; a proliferative/regeneration stage and finally a tissue remodeling phase. Data from the literature suggest that the delayed healing in diabetic patient is principally due to chronic inflammation. Our hypothesis was to demonstrate that deregulation was the result of a major impairment of the resolution phase of inflammation. Our ultimate aim was to influence this phase to accelerate the healing process in diabetic mice. The first step of this study was to develop a new cutaneous excisional wound model in type 2 diabetic mice (in High Fat Diet or leptin receptor deficiency mice db/db). Associated with the development of an innovative device, this model allowed us (i) to treat the wound and follow the wound healing evolution (ii), to collect in aseptically condition and over time the secreted exudate, (ii) to evaluate the phenotypic and functional characteristics of exudate cells and to measure the amount of pro-and anti -inflammatory mediators. This first step leads to the publication of two patent applications (filed in December 2011 and published under the references FR 2984719 and FR 2984722). Our experimental approach demonstrate that the impaired wound healing process in type 2 diabetic mice was due to a strong neutrophil and inflammatory macrophage M1 infiltration, and a lack of influx of anti-inflammatory macrophages M2. All this is associated with a failure in the inflammatory cells apoptosis and efferocytosis. These dysregulations sign a default of resolution of inflammation that we have quantified by the measure of excessive leukotriene (LT) B4, (a proinflammatory arachidonic acid (AA) metabolite from the 5-lipoxygenase pathway) in both exudate and scar tissue, at the expense of the lipoxin (LX) A4 (an anti-inflammatory AA metabolite formed from LTA4 by 12/15-LOX). With an original pharmacological approach, we target the resolution of the inflammatory phase to promote wound closure in diabetic mice. For this we have decided to use topical application of acetylsalicylic acid on the wound (AAS-aspirin), 3 days after the establishment of the skin lesion. We demonstrate that aspirin accelerates wound closure in diabetic mice.

Introduction: Chronic wounds are a great burden to care-givers and patients alike and are the main cause of many preventable amputations. Such wounds are treated with wound dressings but providing a wound environment that is conducive to proper wound healing is not always possible with such dressings. Absorbatox™ is a natural zeolite that has been manipulated to increase its cationic exchange capacity and has its main functionality as a potential wound healing agent in its strong capillary action. This quality enables the zeolite to absorb excess wound exudate and thus prevent wound infection and maceration. Absorbatox™ was characterised to determine its effects on wound healing. Methods: The physical characterisation of two grades of Absorbatox™ - granular and micronised - was conducted using nitrogen adsorption to determine pore size and surface area, and laser particle sizing to determine the particle sizes of the Absorbatox™ particles. Full-thickness wounds of 8 x 8 mm were created on the backs of pigs and treated with Absorbatox™, a positive and a negative control. The wound dimensions were measured and recorded. The wounds were then excised on selected days of each phase of wound healing and fixed in formalin. The wound sections were analysed by mass spectrometry imaging and abundant wound proteins were identified from the tryptic digests using BLAST against the Swiss-Prot database. Results: The surface areas of the micronised and granular Absorbatox™ were 14.43 and 11.23 m2/g, respectively. The micronised Absorbatox™ particle sizes ranged between 0.8 µm to approximately 300 µm with an average pore diameter of 28.2 nm. The granular Absorbatox™ particle sizes ranged between 2 µm and 875 µm with average pore diameters of 43.8 nm. Absorbatox™ showed better wound healing by delaying wound contraction and causing more rapid shallowing of the wound depths compared to the negative control. The difference observed in the wound healing rates of the Absorbatox™-treated and positive control groups were statistically significant and the histological evaluations of the wounds treated with Absorbatox™ showed wound closures that were associated with qualities that more closely resembled normal, healthy tissue than the positive control wounds. The protein activity in the trypsin-digested tissue including within the wound area and the surrounding healthy tissue was successfully imaged using MALDI-MSI. BLAST software was used at an e-value of 30 to identify possible proteins from the tryptic digests and were identified as proteins involved in wound healing. Discussion: Micronised Absorbatox™ treated wounds showed more rapid healing than the other treatments most likely due to the smaller particles and pores which results in strong capillary action to absorb excess exudate. Mass spectrometry imaging allowed monitoring of the protein fluctuations that occur during wound healing. The proteins detected were then identified using BLAST and MASCOT database comparison tools which identified that the abundant proteins detected by mass spectrometry were not those typically observed in wound healing but rather those involved in molecular aspects of wound healing like nerve regeneration, cell proliferation, survival, and migration. / Dissertation (MSc)--University of Pretoria / Pharmacology / unrestricted

Submitted in fulfillment for the Degree of Master of Applied Sciences in Biotechnology, Durban University of Technology, Durban, South Africa, 2017. / South Africa has a wide range of medicinal plants that are used traditionally by black Zulu South Africans for the treatment of a range of illnesses, including inflammatory ailments; disease conditions caused by oxidative stress and wound healing. It has been indicated that bioactive compounds isolated from plants contribute to their anti-inflammatory, antioxidant and wound healing properties; hence, herbal remedies have been widely used traditionally in many countries in the management and treatment of wounds. Inflammation is the main condition that relates to a variety of diseases affecting most of the world’s population. It is the body’s immune response to infection and injury and is induced by the release of pro-inflammatory mediator’s —prostaglandins and leukotrienes—following wound occurrence. Wounds result in disruption of living tissue caused by oxidative stress. Anti-inflammatory agents, antioxidants, and antimicrobials play an important role in the wound healing process and they prevent aggravated wound conditions.Controlling inflammation during wound repair is important to minimize any additional complications that may result; hence, chemical agents such as non-steroidal anti-inflammatory drugs (NSAIDS), synthetic antioxidantsand steroids are frequently used. These drugs block the enzymes that are responsible for prostaglandin synthesis in inflammation, react with free radicals thereby interfering with oxidation process as a result affect one or more phases of wound healing. The use of these drugs, however, has been limited as they can cause detrimental side effects when used over long periods of time.There is, consequently, a need to find alternative natural therapeutic drugs. Studies on medicinal plants confirmed that herbal drugs exhibit fewer side effects in comparison with chemical agents and are more cost-effective.Thus the aim of this study was to investigate South African medicinal plants, for anti-inflammatory, antioxidant and wound healing properties. Dissolved extracts of thirty-eight medicinal plants were evaluated for theiranti-inflammatory activity using the 5-lipoxygenase assay as well as free radical scavenging activity using the 1; 1-diphenyl-2-picrylhydrazyl (DPPH) assay.Their safety was evaluated using brine shrimp lethality assay. Proliferation and viability of fibroblast cells was determined by the3-(4, 5-dimethylthiazolyl)-2, 5-diphenyltetrazolium bromide(MTT) assay furthermore a scratch wound assay was used to study the properties of wound healing in vitro and to confirm the anti-inflammatory activities of the dissolved extracts. Migration rate was evaluated quantitatively by an image analyzer. Methanol was chosen for extraction because it completely dissolves extracts. Anova was used for statistical analysis. Almost all aqueous extracts were found to be effective in inhibiting lipoxygenase enzyme when compared to nordihydroguaiaretic acid (NDGA). Aqueous extracts exhibited remarkably high percentage inhibition of lipoxygenase with most above 100% when compared to methanolic extracts. Amaranthus dubius and Portulaca oleracea were found to have good biological activities in the inhibition of 5-lipoxygenase enzymes when compared to the other plants. However, Galinsoga parviflora and Syzygium cordatumwere least effective in inhibiting enzyme activity with percentages as low as -2% and 34% respectively. Percentage inhibitions for methanolic extracts were lower than that of aqueous extracts. Amaranthus spinosus had the highest percentage inhibition among all the methanolic extracts andGalinsoga parviflorahad the lowest. The methanolic plant extracts were found to be more effective in scavenging DPPH free radicals than the corresponding aqueous extracts. All the methanolic extracts exhibited free radical scavenging ability in the range of 60%–104%. Asystasia gangetica, Ficus sur, Heteropyxis natalensis, Hibiscus sabdariffa, Pelargonium sp. showed notably higher scavenging abilities, ranging from 101%–104% compared to Rutin. Methanolic extracts of Heteropyxis natalensis and Hibiscus sabdariffa exhibited scavenging ability even at the lowest concentration of 10μg/ml. Furthermore, aqueous extracts displayed remarkably lower activities than methanolic extracts with thirty-one extracts having a scavenging capacity ranging from 22%—59%. None of the extracts were found to be detrimental to brine shrimp. Almost all the extracts were shown to stimulate the growth of fibroblast cells except the methanolic extract of Solanum nodiflorum, which was shown to be killing the cells at high concentrations with a percentage viability of 46%.As the concentration decreased, however, the viability of cells with this extract increased to 143%. An increase in the number of fibroblast cells was observed in the scratched area of the treated cells and a significant migration rate was also noted with some of the extracts. Aqueous extracts of Sonchus oleraceus (86%), Justicia flava (85%) and Dichrostachys cinerea (85%) and methanolic extracts of Senna occidentalis and Hibiscus sabdariffa were found to have the highest migration rate compared to untreated cells that served as a control. No cell migration was observed with methanolic extract of Solanum nodiflorum.Instead, the extract was found to be toxic to the cells. Some of the plants evaluated in this study have been studied for either anti-inflammatory, antioxidantand wound healing properties in vivo, however, no work has been conducted to demonstrate a correlation between anti-inflammatory, antioxidant and wound healing properties of plant species in vitro. The current study was, therefore, conducted to review medicinal herbs considered as anti-inflammatory, antioxidants and wound healing agents as well as collecting evidence for their effectiveness and pharmacological mechanisms in modern science. In the plant species investigated Amaranthus dubius, Asystasia gangetica, Bidens pilosa, Buddleja saligna, Carpobrotus dimidiatus, Chenopodium album, Dichrostachys cinerea, Emex australis, Ficus sur, Guilleminea densa, Hibiscus sabdariffa, Physalis viscose, Syzygium cordatum, Taraxacum officinale and Tulbaghia violacea demonstrated good anti-inflammatory and wound healing properties.In conclusion the results from this study demonstrated promising anti-inflammatory and antioxidantactivities as well as wound healing properties,furthermoreit was aslo shown that the plant extracts were not toxic to the cells hencethis suggested that the plants investigated, can be used as substitutes or to formulate wound healing agents that are safe to use in primary healthcare. / M